RESUMO
Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. We have previously demonstrated that the Aggregatibacter actinomycetemcomitans Cdt induces a pro-inflammatory response from human macrophages which involves activation of the NLRP3 inflammasome. We now demonstrate that in addition to activating caspase-1 (canonical inflammasome), Cdt treatment leads to caspase-4 activation and involvement of the noncanonical inflammasome. Cdt-treated cells exhibit pyroptosis characterised by cleavage of gasdermin-D (GSDMD), release of HMGB1 at 24 hr and LDH at 48 hr. Inhibition of either the canonical (caspase-1) or noncanonical (caspase-4) inflammasome blocks both Cdt-induced release of IL-1ß and induction of pyroptosis. Analysis of upstream events indicates that Cdt induces Syk phosphorylation (activation); furthermore, blockade of Syk expression and inhibition of pSyk activity inhibit both Cdt-induced cytokine release and pyroptosis. Finally, we demonstrate that increases in pSyk are dependent upon Cdt-induced activation of GSK3ß. These studies advance our understanding of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as A. actinomycetemcomitans.
Assuntos
Toxinas Bacterianas/efeitos adversos , Glicogênio Sintase Quinase 3 beta/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Quinase Syk/metabolismo , Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Citocinas/metabolismo , Proteína HMGB1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Piroptose , Células THP-1RESUMO
Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger-associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro-inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF-κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase-1 and stimulates secretion of mature IL-1ß. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high-mobility group box 1 protein and apoptosis-associated speck-like protein, with a similar time-course as caspase-1 activation. Thus, F. nucleatum expresses the pathogen-associated molecular patterns necessary to activate NF-κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Infecções por Fusobacterium/metabolismo , Gengiva/microbiologia , Proteína HMGB1/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Fusobacterium/microbiologia , Fusobacterium nucleatum/patogenicidade , Gengiva/citologia , Gengiva/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de SinaisRESUMO
Several lines of evidence suggest that hnRNPs A/B (hnRNPs A1, A2/B1, and A3) play an important role in proliferation, although the functional overlap among members of hnRNPs A/B remains largely unknown. In this study, we have employed RNAi knockdown and proteomic approaches to investigate the biological functions of hnRNPs A/B. Depletion of hnRNP A2, but not A1 or A3, produced a significant inhibition of cellular proliferation in Jurkat cells. Analysis of the proteomes in the cells depleted for hnRNP A1, A2, or A3 has identified a total of 167 differentially expressed proteins in the depleted cells. Network analysis of the proteins altered in the cells depleted for hnRNP A2 revealed that the biological processes likely affected by these proteins are related to cell cycle, cytoskeleton rearrangement, and transcription regulation. Indeed, we have confirmed that the level of RhoA and CrkL was selectively reduced in the cells depleted of hnRNP A2, but not in the cells depleted for hnRNP A1 or A3. Therefore, we suggest that the reduced proliferation observed in the cells depleted of hnRNP A2 may result from its effects on cell adhesion processes in the Jurkat cells.
Assuntos
Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Células Jurkat/metabolismo , Proteoma/metabolismo , Proliferação de Células , Desenho de Equipamento , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Células Jurkat/citologia , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Proteoma/análise , Proteoma/genética , Proteômica/instrumentação , Proteômica/métodos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Agents that interact selectively with telomerase are anticipated to exert specific action on cancer cells. In this study, we evaluated maleimide derivatives for their potency and selectivity of telomerase inhibition. Among the several N-substituted derivatives of maleimide tested, N-(1-Pyrenyl) maleimide was shown to exert the greatest inhibition of telomerase in a cell free system, with an IC50 value of 0.25 µM. Importantly, we demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat T cells and displays the greatest differential cytotoxicity against hematopoietic cancer cells. These results suggest that N-(1-pyrenyl) maleimide is an attractive maleimide to be tested and developed as anti-cancer drug.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Maleimidas/farmacologia , Telomerase/antagonistas & inibidores , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Humanos , Concentração Inibidora 50 , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Maleimidas/química , Maleimidas/toxicidadeRESUMO
Telomeres are dynamic DNA-protein complexes that protect the ends of linear chromosome. Telomere-binding proteins play crucial role in the maintenance of telomeres. HnRNP A3 has been shown recently to bind specifically to single-stranded telomeric DNA in vitro, although its in vivo telomere function remains unknown. In this study, the DNA-binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. The minimal sequence for hnRNP A3 binding to DNA was determined as an undecamer with the following consensus sequence 5'-[T/C]AG[G/T]NN[T/C]AG[G/T]N-3'. Confocal microscopy and chromatin-immunoprecipitation (ChIP) analyses showed that hnRNP A3 is associated with telomere in vivo. Knocking-down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation. In contrast, overexpression of hnRNP A3 resulted in the production of steady-state short telomeres in OECM1 cells. These results suggest that hnRNP A3 is associated with telomere in vivo and acts as a negative regulator of telomere length maintenance.
Assuntos
DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Hep G2 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Microscopia Confocal , Mutação , Ligação Proteica , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Telômero/genéticaRESUMO
BACKGROUND: The formation of dental plaque and its involvement in the pathogenesis of periodontitis is a topic of intense interest given the high prevalence of periodontitis in humans. Even though calcium-based particles play an active role in both dental plaque formation and periodontitis, few publications describe the physical-chemical properties of these particles. METHODS: Saliva samples were collected from healthy volunteers. From these samples, saliva-derived particles were isolated and stained for calcium using calcein or Fluo-4. The salivary particles were also subjected to characterization by flow cytometry and immunoblotting. Internalization of calcein-labeled salivary particles by gingival epithelial cells was visualized by confocal microscopy. RESULTS: We found that calcium-based salivary particles from healthy volunteers varied greatly in size but were enriched in particles of sizes at or greater than 1.5 µm. Immunoblotting analysis of the salivary particles identified several proteins including albumin, fetuin-A, and statherin, which have been found in calcium phosphate particles from other tissues or are known to modulate calcium homeostasis in saliva. In addition, calcium particles were internalized by both gingival epithelial cells and monocyte-derived macrophages. CONCLUSION: Salivary calcium particles were enriched in the micrometer range, internalized by gingival epithelial cells, and contain albumin, fetuin-A and statherin, regulators of particle formation. These characteristics of the calcium-based salivary particles and their biological activities provide a basis for further studies to understand the molecular basis for pathogenesis of periodontitis.
Assuntos
Cálcio , Periodontite , Cálcio/análise , Cálcio/metabolismo , Epitélio/metabolismo , Gengiva/metabolismo , Gengiva/patologia , Humanos , Saliva/químicaRESUMO
Telomeres are dynamic DNA-protein complexes at the end of linear chromosomes. Maintenance of functional telomeres is required for chromosome stability, and to avoid the activation of DNA damage response pathway and cell cycle arrest. Telomere-binding proteins play crucial roles in the maintenance of functional telomeres. In this study, we employed affinity pull-down and proteomic approach to search for novel proteins that interact with the single-stranded telomeric DNA. The proteins identified by two-dimensional gel electrophoresis were further characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF-TOF tandem MS. Among the five identified proteins, we report here the biochemical properties of a novel protein, hnRNP A3. The purified hnRNP A3 bound specifically to G-rich strand, but not to C-rich strand or double-stranded telomeric DNA. The RRM1 (RNA recognition motif 1) domain, but not RRM2, of hnRNP A3 is sufficient to confer specific binding to the telomeric sequence. In addition, we present evidence that hnRNP A3 can inhibit telomerase extension in vitro. These biochemical properties of hnRNP A3 suggest that hnRNP A3 can participate in telomere regulation in vivo.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Telomerase/antagonistas & inibidores , Telômero/metabolismo , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Clonagem Molecular , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/isolamento & purificação , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The A/B subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs A/B), which includes hnRNP A1, A2/B1, and A3, plays an important role in cell proliferation. The simultaneous suppression of hnRNP A1/A2, but not the suppression of hnRNP A1 or A2 alone, has been shown to inhibit cell proliferation and induce apoptosis in cancer cells, but not in mortal normal cells. However, the molecular basis for such a differential inhibition of cell proliferation remains unknown. Here, we show that the simultaneous suppression of hnRNP A1 and hnRNP A2 resulted in dysfunctional telomeres and induced DNA damage responses in cancer cells. The inhibition of apoptosis did not alleviate the inhibition of cell proliferation nor the formation of dysfunctional telomeres in cancer cells depleted of hnRNP A1/A2. Moreover, while proliferation of mortal normal fibroblasts was not sensitive to the depletion of hnRNP A1/A2, the ectopic expression of hTERT in normal fibroblasts rendered these cells sensitive to proliferation inhibition, which was associated with the production of dysfunctional telomeres. Our study demonstrates that hnRNP A1 and A2 function to maintain telomeres in telomerase-expressing cells only, suggesting that the maintenance of functional telomeres in telomerase-expressing cancer cells employs factors that differ from those used in the telomerase-negative normal cells.
RESUMO
Chronic psychostimulant treatment in rodents readily produces behavioral sensitization, which reflects altered brain function in response to repeated drug exposure. Numerous morphological and biochemical investigations implicate altered neural plasticity in striatal medium spiny neurons (MSNs) as an essential component in behavioral sensitization. The mammalian target of the rapamycin (mTOR) signaling pathway, a key regulator of synaptic neuroplasticity, in the ventral striatum of methamphetamine (METH) -sensitized mice was investigated to determine if a link exists with the development of METH sensitization. Behaviorally, METH-sensitized mice possessed increased levels of phosphorylated mTOR/S2448 and its down-stream regulator p70S6K and pS6 in the ventral striatum. Systemic treatment with rapamycin, a specific mTOR inhibitor, coincident with a daily METH injection suppressed the induction of METH sensitization and reduced the number of dendritic spines in the shell and core of the nucleus accumbens. The infusion of lentivirus-expressing mTOR-shRNA into the shell region of the nucleus accumbens inhibited the induction of behavioral sensitization to METH, which was comparable to the effect of rapamycin. These results suggest that mTORC1-mediated signaling in the nucleus accumbens mediates the development of behavioral sensitization to METH.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Metanfetamina/farmacologia , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Núcleo Accumbens/metabolismo , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
NOD-like receptors (NLRs) play a large role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRX1, a member of the NLR family that localizes to mitochondria, enhances mitochondrial ROS (mROS) generation. mROS can activate the NLRP3 inflammasome, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we revealed the mechanism by which NLRX1 positively regulates ATP-induced NLRP3 inflammasome activation through mROS in gingival epithelial cells (GECs). We found that depletion of NLRX1 by shRNA attenuated ATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, depletion of NLRX1 inhibited Fusobacterium nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Conversely, NLRX1 also acted as a negative regulator of NF-κB signaling and IL-8 expression. Thus, NLRX1 stimulates detection of the pathogen F. nucleatum via the inflammasome, while dampening cytokine production. We expect that commensals should not activate the inflammasome, and NLRX1 should decrease their ability to stimulate expression of pro-inflammatory cytokines such as IL-8. Therefore, NLRX1 may act as a potential switch with regards to anti-microbial responses in healthy or diseased states in the oral cavity.
Assuntos
Infecções por Fusobacterium/metabolismo , Inflamassomos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Fusobacterium nucleatum/fisiologia , Expressão Gênica , Gengiva , Humanos , Interleucina-8/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismoRESUMO
Although human blood is believed to be a sterile environment, recent studies suggest that pleomorphic bacteria exist in the blood of healthy humans. These studies have led to the development of "live-blood analysis," a technique used by alternative medicine practitioners to diagnose various human conditions, including allergies, cancer, cardiovascular disease and septicemia. We show here that bacteria-like vesicles and refringent particles form in healthy human blood observed under dark-field microscopy. These structures gradually increase in number during incubation and show morphologies reminiscent of cells undergoing division. Based on lipid analysis and Western blotting, we show that the bacteria-like entities consist of membrane vesicles containing serum and exosome proteins, including albumin, fetuin-A, apolipoprotein-A1, alkaline phosphatase, TNFR1 and CD63. In contrast, the refringent particles represent protein aggregates that contain several blood proteins. 16S rDNA PCR analysis reveals the presence of bacterial DNA in incubated blood samples but also in negative controls, indicating that the amplified sequences represent contaminants. These results suggest that the bacteria-like vesicles and refringent particles observed in human blood represent non-living membrane vesicles and protein aggregates derived from blood. The phenomena observed during live-blood analysis are therefore consistent with time-dependent decay of cells and body fluids during incubation ex vivo.
Assuntos
Proteínas Sanguíneas , Vesículas Extracelulares/metabolismo , Proteínas/metabolismo , Biomarcadores , Difusão Dinâmica da Luz , Vesículas Extracelulares/ultraestrutura , Humanos , Lipídeos/sangue , Microscopia , Agregados Proteicos , RNA Ribossômico 16S/genética , Análise EspectralRESUMO
Calcium phosphate-based mineralo-organic particles form spontaneously in the body and may represent precursors of ectopic calcification. We have shown earlier that these particles induce activation of caspase-1 and secretion of IL-1ß by macrophages. However, whether the particles may produce other effects on immune cells is unclear. Here, we show that these particles induce the release of neutrophil extracellular traps (NETs) in a size-dependent manner by human neutrophils. Intracellular production of reactive oxygen species is required for particle-induced NET release by neutrophils. NETs contain the high-mobility group protein B1 (HMGB1), a DNA-binding protein capable of inducing secretion of TNF-α by a monocyte/macrophage cell line and primary macrophages. HMGB1 functions as a ligand of Toll-like receptors 2 and 4 on macrophages, leading to activation of the MyD88 pathway and TNF-α production. Furthermore, HMGB1 is critical to activate the particle-induced pro-inflammatory cascade in the peritoneum of mice. These results indicate that mineral particles promote pro-inflammatory responses by engaging neutrophils and macrophages via signaling of danger signals through NETs.
Assuntos
Armadilhas Extracelulares/imunologia , Proteína HMGB1/metabolismo , Imunidade Inata , Imunomodulação , Minerais/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Animais , Linhagem Celular , Feminino , Proteína HMGB1/genética , Humanos , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Modelos Moleculares , Fator 88 de Diferenciação Mieloide/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Inhibition of telomerase in cancer cells has been shown to limit the growth of human cancer cells in culture. In this study, we report that helenalin, a natural sesquiterpene lactone, is a potent and selective inhibitor for human telomerase. In vitro studies indicate that this drug can inactivate telomerase directly in a manner that is dependent on concentration and time. The inhibitory action of this drug on telomerase is selective since the presence of excessive externally added proteins did not protect the inhibition and all of the other enzymes tested in this study were not inhibited by this drug. Furthermore, we demonstrated that helenalin can inhibit the expression of hTERT and telomerase in hematopoietic cancer cells. Therefore, the anti-tumor activity of helenalin is attributed, at least in part, to the inhibition of telomerase.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Sesquiterpenos/farmacologia , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Células HL-60/enzimologia , Humanos , Técnicas In Vitro , Células Jurkat/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sesquiterpenos de Guaiano , Telomerase/genética , Toxinas BiológicasRESUMO
N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.
Assuntos
Maleimidas/farmacologia , Mitocôndrias/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Proteína X Associada a bcl-2/metabolismoRESUMO
A one-step process for the synthesis of hydrophilic carbon nanofibers (CNFs) through CO2 hydrogenation on NiNa/Al2O3 was developed for the loading and targeted delivery of the anticancer drug doxorubicin (DOX). CNFs that were synthesized on NiNa/Al2O3 for 9 h at 500 °C exhibited an adequate magnetic response and a large content of hydrophilic oxygen-containing functional groups on the carbon surface, resulting in excellent colloidal solution. The CNF material exhibited a highly efficient capacity for DOX adsorption, particularly at pH 9.0. The loading and release of DOX was strongly pH dependent, possibly due to electrostatic and π-π stacking interactions between DOX and CNF sample. The Langmuir isotherm and pseudo second-order kinetics of DOX-loaded CNFs were well-modeled for the process of DOX adsorption. DOX-loaded CNF targeted cancer cells more selectively and effectively than free DOX and exhibited a marked tendency to kill HeLa cancer cells and reduced toxicity to normal human primary fibroblast (HPF) cells.
Assuntos
Carbono/química , Sistemas de Liberação de Medicamentos , Nanopartículas Metálicas/química , Nanofibras/química , Níquel/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Espectrofotometria InfravermelhoRESUMO
Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC(50) value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 microM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC(50) values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.